The present invention generally relates to the SCARECROW (SCR) gene family and their promoters. The invention more particularly relates to ectopic expression of members of the SCARECROW gene family in transgenic plants to artificially modify plant structures. The invention also relates to utilization of the SCARECROW promoter for tissue and organ specific expression of heterologous gene products.
Asymmetric cell divisions, in which a cell divides to give two daughters with different fates, play an important role in the development of all multicellular organisms. In plants, because there is no cell migration, the regulation of asymmetric cell divisions is of heightened importance in determining organ morphology. In contrast to animal embryogenesis, most plant organs are not formed during embryogenesis. Rather, cells that form the apical meristems are set aside at the shoot and root poles. These reservoirs of stem cells are considered to be the source of all post-embryonic organ development in plants. A fundamental question in developmental biology is how meristems function to generate plant organs.
2.1. Root Development
Root organization is established during embryogenesis. This organization is propagated during postembryonic development by the root meristem. Following germination, the development of the postembryonic root is a continuous process, wherein a series of initials or stem cells continuously divide to perpetuate the pattern established in the embryonic root (Steeves and Sussex, 1972, Patterns in Plant Development, Englewood Cliffs, N.J.: Prentice-Hall, Inc.).
2.1.1. Arabidopsis Root Development
Due to the organization of the Arabidopsis root, it is possible to follow the fate of cells from the meristem to maturity and identify the progenitors of each cell type (Dolan et al., 1993, Development 119:71-84). The Arabidopsis root is a relatively simple and well characterized organ. The radial organization of the mature tissues in the Arabidopsis root has been likened to tree rings with the epidermis, cortex, endodermis and pericycle forming radially symmetric cell layers that surround the vascular cylinder (FIG. 1A). See also Dolan et al., 1993, Development 119:71-84. These mature tissues are derived from four sets of stem cells or initials: i) the columella root cap initial; ii) the pericycle/vascular initial; iii) the epidermal/lateral root cap initial; and iv) the cortex/endodermal initial (Dolan et al., 1993, Development 119:71-84). It has been""shown that these initials undergo asymmetric divisions (Scheres et al., 1995, Development 121:53-62). The cortex/endodermal initial, for example, first divides anticlinally (in a transverse orientation) (FIG. 1B). This asymmetric division produces another initial and a daughter cell. The daughter cell, in turn, expands and then divides periclinally (in the longitudinal orientation) (FIG. 1B). This second asymmetric division produces the progenitors of the endodermis and the cortex cell lineages (FIG. 1B).
Furthermore, root radial organization in Arabidopsis is produced by three distinct developmental strategies. First, primary roots employ stem cells, wherein initials undergo asymmetric divisions first to regenerate themselves and then to generate the cell lineages of the root (FIG. 1B). Second, in the embryo, sequential asymmetric divisions subdivide pre-existing tissue to form the cell layers of the embryonic root. Finally, lateral roots are formed by a strategy of cell proliferation that originates in differentiated tissues. Remarkably, within a given species, all three strategies result in roots with a nearly identical radial organization.
2.1.2. Maize Root Development
The root organization of Zea mays (maize), which is a very well characterized member of the grass family, is far more complex than the root organization in Arabidopsis. The root system of maize consists of primary, embryonic, lateral, seminal lateral and adventitious roots. Both primary and seminal lateral roots are formed during embryogenesis, wherein the primary root is the first root to emerge during germination, followed by the seminal lateral roots formed at the scutellar nodal region (Freeling, M. and Walbot, V. (1994), The Maize Handbook, (New York: Springer-Verlag); Hetz, W. et al., (1996), Plant J. 10:845-857). Both crown and prop roots which develop post-embryonically are shoot-borne roots, often termed xe2x80x9cadventitiousxe2x80x9d. However, since these roots are part of the normal development of the plant, they are not, strictly speaking, adventitious roots, which are typically formed as a result of injury or hormone treatment. Crown roots, representing the major roots of the mature plant, are formed at consecutive early nodes of the stem beginning with the coleoptilar nodes. Later in development, brace or prop roots emerge from nodes above the soil level (Freeling, M. and Walbot, V. (1994), The Maize Handbook, (New York: Springer-Verlag); Hetz, W. et al., (1996), Plant J. 10:845-857).
Currently, there are two notably different types of organization of root apical meristems: an open and a closed meristem. In an xe2x80x9copenxe2x80x9d meristem, the cell files of the mature tissues cannot be traced with much confidence to distinct initials, and the incipient tissues do not appear to have discrete boundary walls between the root proper and the root cap (Clowes, F. A. L., 1981, Ann. Bot. 48:761-767). Therefore, the interpretation of the organization of the open meristem has been problematic (Clowes, F. A. L., 1981, Ann. Bot. 48:761-767). In a xe2x80x9cclosedxe2x80x9d meristem, however, since files of cells converge onto a pole at the root apex, it is easy to identify discrete layers in median longitudinal sections (Clowes, F. A. L., 1981, Ann. Bot. 48:761-767).
Both Arabidopsis and maize roots show characteristics of the closed meristem (FIGS. 23A-B). However, there are important differences. In maize roots, the root apical meristem consists of three independent layers of initials. One gives rise to the stele, the second gives rise to epidermis, cortex and endodermis and the third generates the root cap, whereas in the Arabidopsis root apical meristem, the epidermis shares a common initial with the lateral root cap (Esau, K., 1977, Anatomy of Seed Plants. 2nd ed. (New York: John Wiley and Sons); Esau, K., 1953, Plant Anatomy. (New York: John Wiley and Sons)).
Primary organization of the root apical meristem in maize occurs during embryogenesis, (Steeves, T. A. and Sussex, I. M., (1989), Patterns in plant development., 2nd ed. (Cambridge University Press)) as in Arabidopsis. There are three main phases in embryo development in maize (FIGS. 24A-B) (Freeling, M. and Walbot, V. (1994), The Maize Handbook, (New York: Springer-Verlag); Steeves, T. A. and Sussex, I. M. (1989), Patterns in plant development., 2nd ed., (Cambridge University Press); Sheridan, W. F. and Clark, J. K., (1993), Plant J. 3:347-358). As in Arabidops is, the very first division of the zygote establishes the initial asymmetry of the embryo (FIG. 24A). However, unlike Arabidopsis, embryonic development in maize is characterized by rather irregular cell divisions (Sheridan, W. F. and Clark, J. K., (1993), Plant J. 3:347-358). During the first phase, the apical-basal asymmetry of the embryo is established, and the embryo is regionalized into suspensor and embryo proper (FIGS. 24B-C). During the second phase, radial asymmetry appears and the embryonic axis and meristems are established (FIGS. 24D-E) (Clowes, F. A. L., (1978), New Phytol. 80:409-419). Finally, during the third phase, vegetative structures such as embryonic roots and leaves are elaborated (FIGS. 24F-G) (Sheridan, W. F. and Clark, J. K., (1993), Plant J. 3:347-358).
2.1.3. The Quiescent Center
The quiescent center (QC) of root apical meristems of angiosperms is a population of mitotically inactive cells. In the QC of the primary root of maize, for example, the average duration of a mitotic cycle is about 200 hours compared with only 12 hours in the cells just below the QC and 28 hours in the cells just above the QC (Clowes, F. A. L., (1961), J. Exp. Bot. 9:229-238). Moreover, there are also reductions in the rates of synthesis of DNA and protein, and corresponding reductions in the amounts of DNA and RNA per cell (Clowes, F. A. L., (1956), New Phytol. 55:29-34).
Although the precise role of the QC has remained speculative, it is generally accepted that cells within the QC are undifferentiated and, other than the anatomical pattern of cell files, lacking in radial pattern information. This theory has been supported by ablation studies performed in Arabidopsis, wherein, complete laser ablation of the four central cells in the Arabidopsis QC led to subsequent restoration of the QC by cells of the stele. Furthermore, laser ablation of only one or two cells in the QC resulted in differentiation of surrounding initial cells. Analysis of the hobbit mutants further supports these observations. In the hobbit mutants, there is no functional QC, leading all cortex initials to divide into cortex and endodermis during embryogenesis (van den Berg, C., et al., (1995), Nature 378:62-65). Taken together, it is suggested that the QC suppresses differentiation of surrounding initials in the range of a single cell (van den Berg, C., et al., (1995), Nature 378:62-65).
In maize, on which the contemporary view of the role of the QC is based (Feldman, L. J., (1984), Amer. J. Bot. 71:1308-1314; Freeling, M. and Walbot, V., (1994), The maize handbook (New York: Springer-Verlag)), surgical and tissue culture systems were developed to study the organization process of root apical meristems (Feldman, L. J., (1976), Planta 128:207-212). Following removal of the QC, the remaining root regenerates a new root tip. This process appears to involve de novo organization of the QC and the apical meristem (Feldman, L. J., (1976), Planta 128:207-212). In addition, the excised QC itself is capable of generating a new root (Feldman, L. J. and Torrey, J. G., (1976), Amer. J. Bot. 63:345-355). This suggests that there is indeed sufficient radial pattern information in the QC to allow the regeneration of more or less intact roots.
2.2. Genes Regulating Root Structure
Mutations that disrupt the asymmetric divisions of the cortex/endodermal initial have been identified and characterized (Benfey et al., 1993, Development 119:57-70; Scheres et al., 1995, Development 121:53-62). short-root (shr) and scarecrow (scr) mutants are missing a cell layer between the epidermis and the pericycle. In both types of mutants, the cortex/endodermal initial divides anticlinally, but the subsequent periclinal division that increases the number of cell layers does not take place (Benfey et al., 1993, Development 119:57-70; Scheres et al., 1995, Development 121:53-62). The defect is first apparent in the embryo and it extends throughout the entire embryonic axis, which includes the embryonic root and hypocotyl (Scheres et al., 1995, Development 121:53-62). This is true also for other radial organization mutants characterized to date, suggesting that radial patterning that occurs during embryonic development may influence the post-embryonic pattern generated by the meristematic initials (Scheres et al., 1995, Development 121:53-62).
Characterization of the mutant cell layer in shr indicated that two endodermal-specific markers were absent (Benfey et al., 1993, Development 119:57-70). This provided evidence that the wild-type SHR gene may be involved in the specification of endodermis identity.
2.3. Geotropism
In plants, the capacity for gravitropism has been correlated with the presence of amyloplast sedimentation. See, e.g., Volkmann and Sievers, 1979, Encyclopedia Plant Physiol., N.S. vol 7, pp. 573-600; Sack, 1991, Intern. Rev. Cytol. 127:193-252; Bjxc3x6rkmann, 1992, Adv. Space Res. 12:195-201; Poff et al., in The Physiology of Tropisms, Meyerowitz and Somerville (eds); Cold Spring Harbor Laboratory Press, Plainview, N.Y. (1994) pp. 639-664; Barlow, 1995, Plant Cell Environ. 18:951-962. Amyloplast sedimentation only occurs in cells in specific locations at distinct developmental stages. That is, when and where sedimentation occurs is precisely regulated (Sack, 1991, Intern. Rev. Cytol. 127:193-252). In roots, amyloplast sedimentation only occurs in the central (columella) cells of the rootcap; as these cells mature into peripheral cap cells, the amyloplasts no longer sediment (Sack and Kiss, 1989, Amer. J. Bot. 76:454-464; Sievers and Braun, in The Root Cap: Structure and Function, Wassail et al. (eds.), New York: M. Dekker (1996) pp. 31-49). In stems of many plants, including Arabidopsis, amyloplast sedimentation occurs in the starch sheath (endodermis) especially in elongating regions of the stem (von Guttenberg, Die Physioloaischen Scheiden, Handbuch der Pflanzenanatomie; K. Linsbauer (ed.), Berlin: Gebruder Borntraeger, vol. 5 (1943) p. 217; Sack, 1987, Can. J. Bot. 65:1514-1519; Sack, 1991, Intern. Rev. Cytol. 127:193-252; Caspar and Pickard, 1989, Planta 177:185-197; Volkmann et al., 1993, J. Pl. Physiol. 142:710-6).
Gravitropic mutants have been studied for evidence that proves the role of amyloplast sedimentation in gravity sensing. However, many gravitropic mutations affect downstream events such as auxin sensitivity or metabolism (Masson, 1995, BioEssays 17:119-127). Other mutations seem to affect gene products that process information from gravity sensing. For example, the lazy mutants of higher plants and comparable mutants in mosses can clearly sense and respond to gravity, but the mutations reverse the normal polarity of the gravitropic response (Gaiser and Lomax, 1993, Plant Physiol. 102:339-344; Jenkins et al., 1986, Plant Cell Environ 9:637-644). Other mutations appear to affect gravitropism of specific organs. For example, sgr mutants have defective shoot gravitropism (Fukaki et al., 1996, Plant Physiol. 110:933-943; Fukaki et al., 1996, Plant Physiol. 110:945-955; Fukaki et al., 1996, Plant Res. 109:129-137).
Citation or identification of any reference herein shall not be construed as an admission that such reference is available as prior art to the present invention.
The structure and function of a regulatory gene, SCARECROW (SCR), is described. The SCR gene is expressed specifically in root progenitor tissues of embryos, and in certain tissues of roots and stems. SCR expression controls cell division of certain cell types in roots, and affects the organization of root and stem. The present invention relates to the SCARECROW (SCR) gene (which encompasses the Arabidopsis SCR gene and its orthologs and paralogs), SCR-like genes, SCR gene products, (including, but not limited to, transcriptional products such as mRNAs, antisense and ribozyme molecules, and translational products such as the SCR protein, polypeptides, peptides and fusion proteins related thereto), antibodies to SCR gene products, SCR regulatory regions and the use of the foregoing to improve agronomically valuable plants.
The invention is based, in part, on the discovery, identification and cloning of the gene responsible for the scarecrow phenotype. In contrast to the prevailing view that the SCR gene was likely to be involved in the specification of endodermis, the inventors have determined that the mutant cell layer in roots of scr mutants has differentiated characteristics of both cortex and endodermis. This is consistent with a role for SCR in the regulation of asymmetric cell division rather than in specification of the identity of either cortex or endodermis. The inventors have determined also that SCR expression affects the gravitropism of plant aerial structures such as the stem.
One aspect of the invention relates to the heterologous expression of SCR genes and related nucleotide sequences, and specifically the Arabidopsis SCR and maize ZCARECROW (ZCR) genes, in stably transformed higher plant species. Modulation of SCR and ZCR expression levels can be used to advantageously modify root and aerial structures of transgenic plants and enhance the agronomic properties of such plants.
Another aspect of the invention relates to the use of promoters of SCR genes, and specifically the use of the Arabidopsis SCR and maize ZCR promoters to control the expression of protein and RNA products in plants. Plant SCR promoters have a variety of uses, including, but not limited to, expressing heterologous genes in the embryo, root, root nodule and stem of transformed plants.
The invention is illustrated by working examples, described infra, which demonstrate the isolation of the Arabidopsis SCR gene using insertion mutagenesis. More specifically, T-DNA tagging of genomic and cDNA clones of the Arabidopsis SCR gene are described. Other working examples include the isolation of SCR sequences from plant genomes using PCR amplification in combination with screening of genomic libraries, and heterologous gene expression in transgenic plants using SCR promoter expression constructs. Additional working examples describe the cloning and isolation of maize ZCR genes using probes derived from the Arabidopsis SCR gene on a maize genomic library. Still other working examples describe the characterization of the maize ZCR expression pattern in primary and embryonic roots, and during regeneration of the root tip following excision of the QC.
Structural analysis of the deduced amino acid sequence of Arabidopsis SCR protein indicates that SCR encodes a transcription factor. Northern analysis, in situ hybridization analysis and enhancer trap analysis show highly localized expression of Arabidopsis SCR and maize ZCR in embryos and roots. Genetic analysis shows SCR expression also affects gravitropism of aerial structures (e.g., stems and shoots). This indicates that SCR is also expressed in those structures.
Computer analysis of the deduced amino acid sequence of Arabidopsis SCR protein with those of Expressed Sequence Tag (EST) sequences and genomic sequences in GenBank reveals the existence of at least eighteen SCR genes in Arabidopsis, one SCR gene in maize, four SCR genes in rice, and one SCR gene in Brassica. A further aspect of the invention relates to the use of such EST sequences to obtain larger and/or complete clones of the corresponding SCR gene.
The various embodiments of the claimed invention presented herein are by way of illustration only and are in no manner intended to limit the scope of the invention.
3.1. Definitions
As used herein, the terms listed below will have the meanings indicated.
SCR protein means a protein containing sequences or a domain substantially similar to one or more motifs (i.e., Motifs I-VI), preferably MOTIF III (VHIID), of the Arabidopsis SCR protein as shown in FIGS. 13A-F and FIGS. 15A-S. SCR proteins include SCR ortholog and paralog proteins having the structure and activities described herein.
SCR polypeptides and peptides include deleted or truncated forms of the SCR protein, and fragments corresponding to the SCR motifs described herein.
SCR fusion proteins encompass proteins in which the SCR protein or an SCR polypeptide or peptide is fused to a heterologous protein, polypeptide or peptide.
SCR gene, nucleotides or coding sequences mean nucleotides, e.g., gDNA or cDNA encoding SCR protein, SCR polypeptides, peptides or SCR fusion proteins.
SCR gene products include transcriptional products such as mRNAs, antisense and ribozyme molecules, as well as translational products of the SCR nucleotides described herein, including, but not limited to, the SCR protein, polypeptides, peptides and/or SCR fusion proteins.
SCR promoter means the regulatory region native to the SCR gene in a variety of species, which promotes the organ and tissue specific pattern of SCR expression described herein.